1. Field of the Invention
This invention relates to the field of proteoglycans and of cell surface receptors for biological effector molecules, more particularly the use of genetic engineering to define a class of proteoglycans and their constituent functional domains, particularly their glycosaminoglycan attachment regions. The invention includes the use of recombinant DNA vectors to produce proteins in prokaryotic cells and proteoglycans in eukaryotic cells, and a variety of techniques to link the functional domains to biological effector molecules, cell surface receptors, drugs, antibodies, diagnostic agents, and components of microorganisms.
2. Description of the Background
The cellular behavior responsible for the development, repair and maintenance of tissues is regulated, in large part, by interactions between cells and components of their microenvironment. These interactions are mediated by cell surface molecules acting as receptors that bind large insoluble matrix molecules, growth factors, enzymes, and other molecules that induce responses which result in changes of cellular phenotype. Several proteins associated with the cell surface can bind these components. These proteins differ in their specificity and affinity and in their mode of association with the cell surface.
The present inventors have studied a lipophilic proteoglycan containing both heparan sulfate and chondroitin sulfate that is found at the surface of mouse mammary epithelial cells and that behaves as a high affinity receptor specific for multiple components of the interstitial matrix. This proteoglycan has been given the name syndecan-1. The proteoglycan binds the epithelial cells via its heparan sulfate chains to collagen types I, III, and V (Koda, J. E., Rapraeger, A., and Bernfield, M., J. Biol. Chem. (1985) 260: 8157–8162), fibronectin (Saunders, S. and Bernfield, M., J. Cell Biol. (1988) 106: 423–430), and thrombospondin. When its extracellular domain (ectodomain) is cross-linked at the cell surface, it associates intracellularly with the actin cytoskeleton, and the isolated proteoglycan binds directly or indirectly to F-actin (Rapraeger, A., and Bernfield, M., J. Biol. Chem. (1985) 260: 4103–4109). Cultured cells shed the ectodomain from their apical surfaces as a nonlipophilic proteoglycan that contains all of the glycosaminoglycan of the intact molecule. Upon suspension of these cells, the extracellular domain is cleaved from the cell surface; the proteoglycan is not replaced while the cells are suspended (Jalkanen, M., Rapraeger, A., Saunders, S., and Bernfield, M., J. Cell Biol. (1987) 105: 30873096). The proteoglycan is mainly on epithelia in mature tissues (Hayashi, K., Hayashi, M., Jalkanen, M., Firestone, J. H., Trelstad, R. L., and Bernfield, M., J. Histochem. Cytochem. (1987) 35: 1079–1088).
Syndecan-1 undergoes substantial regulation; its size, glycosaminoglycan composition and location at the cell surface vary between cell types, and its expression changes during development. The proteoglycan is located exclusively at the basolateral cell surface of simple epithelia but surrounds stratified epithelial cells. At basolateral cell surfaces, it appears to contain two heparan sulfate and two chrondroitin sulfate chains, but where it surrounds cells, it contains only a single heparan sulfate chain and a single small chrondroitin sulfate chain (Sanderson, R. D., and Bernfield, M., Proc. Natl. Acad. Sci. USA (1987) 238: 491–497). In self-renewing epithelial cell populations, such as the epidermis or vagina, the proteoglycan is lost when the cells terminally differentiate (Hayashi, K., Hayashi, M., Boutin, E., Cunha, G. R., Bernfield, M., and Trelstad, R. L., J. Lab. Invest. (1988) 58: 68–76). In embryos, the proteoglycan is transiently lost when epithelia change their shape and is transiently expressed by mesenchymal cells undergoing morphogenetic tissue interaction.
Heparan sulfate proteoglycans are ubiquitous on the surfaces of adherent cells and bind various ligands including extracellular matrix, growth factors, proteinase inhibitors, and lipoprotein lipase; see Fransson, L., Trends Biochem. Sci. (1987) 12: 406411, Bernfield et al. (1992) Annu. Rev. Cell. Biol. 8:365–93 However, despite much study of these molecules, no structure was known for the core protein prior to this invention of any such cell surface proteoglycan.
For general background on genetic engineering, see Watson, J. D., The Molecular Biology of the Gene, 4th Ed., Benjamin, Menlo Park, Calif., (1988).